Rescue of Misrouted GnRHR Mutants Reveals Its Constitutive Activity

Abstract

G protein-coupled receptors (GPCR) play central roles in almost all physiological functions, and mutations in GPCR are responsible for over 30 hereditary diseases associated with loss or gain of receptor function. Gain of function mutants are frequently described as having constitutive activity (CA), that is, they activate effectors in the absence of agonist occupancy. Although many GPCR have mutants with CA, the GnRH receptor (GnRHR) was not, until 2010, associated with any CA mutants. The explanation for the failure to observe CA appears to be that the quality control system of the cell recognizes CA mutants of GnRHR as misfolded and retains them in the endoplasmic reticulum. In the present study, we identified several human (h)GnRHR mutants with substitutions in transmembrane helix 6 (F²⁷²K, F²⁷²Q, Y²⁸⁴F, C²⁷⁹A, and C²⁷⁹S) that demonstrate varying levels of CA after being rescued by pharmacoperones from different chemical classes and/or deletion of residue K¹⁹¹, a modification that increases trafficking to the plasma membrane. The movement of the mutants from the endoplasmic reticulum (unrescued) to the plasma membrane (after rescue) is supported by confocal microscopy. Judging from the receptor-stimulated inositol phosphate production, mutants F²⁷²K and F²⁷²Q, after rescue, display the largest level of CA, an amount that is comparable with agonist-stimulated activation. Because mutations in other GPCR are, like the hGnRHR, scrutinized by the quality control system, this general approach may reveal CA in receptor mutants from other systems. A computer model of the hGnRHR and these mutants was used to evaluate the conformation associated with CA.

GnRH receptors (GnRHR) are G protein-coupled receptors (GPCR) that play a key role in regulating reproduction. Numerous point mutations, associated with the disease hypogonadotropic hypogonadism, were found in human GnRHR (hGnRHR) (1). These mutations are widely distributed over the receptor structure, including its N terminus (N¹⁰K, Q¹¹K, and T³²I), transmembrane helix (TM)2 (E⁹⁰K), extracellular loop (EL)1 (Y¹⁰⁴I, Q¹⁰⁶R, and Y¹⁰⁸C), TM3 (A¹²⁹D and R¹³⁹H), TM4 (A¹⁷¹T and S¹⁶⁸R), EL2 (C²⁰⁰Y), TM5 (S²¹⁷R), TM6 (R²⁶²Q, L²⁶⁶R, C²⁷⁹Y, and Y²⁸⁴C), and TM7 (L³¹⁴X_stop and P³²⁰L). Most of these naturally occurring, as well as genetically engineered, mutants are misfolded and misrouted molecules (1, 2) that are retained in the endoplasmic reticulum (ER) (2). This has been shown by relief of the dominant negative effect, whereby misfolded mutants oligomerize with wild type (WT) and retain it in the ER, an effect that is reversed by pharmacoperone drugs (2). Additionally, in the case of two hGnRHR mutants labeled with green fluorescent protein (GFP), E⁹⁰K, and D⁹⁸N, confocal microscopy shows that these are ER retained and, after treatment with pharmacoperones, traffic to the plasma membrane (3, 4).

Pharmacoperone drugs are small molecules that enter cells and act as templates to allow correct folding (or refolding) of mutants. Correctly folded mutants traffic to the plasma membrane and show biological activity (1, 3, 5–8). The WT hGnRHR has a less than perfect chance of folding correctly. This is due to steric inhibition by residue K¹⁹¹ of the formation of the Cys¹⁴-Cys²⁰⁰ bridge. Deletion of K¹⁹¹ or the use of pharmacoperones increases the plasma membrane expression of WT hGnRHR (9, 10) by stabilizing the conformation that is acceptable to the cellular quality control system (QCS). Because the WT hGnRHR is balanced between the plasma membrane and the ER, the human receptor is highly susceptible to misrouting due to single point mutations (11).

The biochemical mechanism by which pharmacoperones engage and rescue mutants has been described (3, 4, 12). For the four different chemical classes of these drugs used in the present work (In3, Q89, A177775, and TAK-013), the drug creates a surrogate bridge between residues D⁹⁸ and K¹²¹, stabilizing a relation between transmembrane segments 2 and 3, which is required for trafficking to the plasma membrane. The biochemical mechanism by which altered residues cause ER retention has been summarized for several misfolded mutants (13, 14). Because the efficiency of recognition by the cellular QCS differs, ER retention is different.

Among several hundreds of GnRHR mutants reported before 2010, none were known to show constitutive activity (CA) (15). This observation was surprising, because most GPCR have multiple mutants with CA (16). The absence of such proteins has been one of the impediments to understanding the active conformation of the GnRHR.

The first mutant identified with CA was E⁹⁰K, which has been extensively studied in our laboratory (1, 3–5, 10, 17–19). This naturally occurring mutation, identified in patients with hypogonadotropic hypogonadism, affects a highly conserved structural feature of the GnRHR, a salt bridge between TM2 and TM3 (4), created by residues E⁹⁰ and K¹²¹ in the human receptor sequence (20, 21). GnRH agonists, but not antagonists, bind both receptor residues D⁹⁸ and K¹²¹ (20, 21) and interfere with the E⁹⁰-K¹²¹ salt bridge, altering the physical relation between TM2 and TM3. Formation of this bridge is required for trafficking of the hGnRHR to the plasma membrane (4), and accordingly, this mutant is 100% retained in the ER and 100% rescuable by pharmacoperones (1, 3–5, 19).

We predicted that mutant E⁹⁰K would result in charge repulsion between K⁹⁰ and K¹²¹ and potentially lead to CA by breaking this salt bridge, as does the agonist. From confocal studies, we also knew that mutant E⁹⁰K is recognized by the QCS as a misfolded protein (22), fully retained in the ER and unable to traffic to the plasma membrane (2). These same studies showed that pharmacoperone drugs rescue this mutant (4) and can, in fact, refold and rescue mutants previously retained in the ER (3). In accordance with this prediction, E⁹⁰K mutant overexpressed in Cos-7 cells clearly demonstrated CA but only after treatment by pharmacoperones or by an additional genetic modification (deletion of residue K¹⁹¹), which increases receptor trafficking of hGnRHR to the plasma membrane (10, 19, 23). Unfortunately, the hGnRHR is among GPCRs, for which it has not been possible to generate antisera, so immunohistochemical localization of the WT or mutant forms of this receptor has not been possible.

In the present study, we suggested that mutations in regions that undergo conformational rearrangement during receptor activation may lead to CA. Therefore, we analyzed functional properties of mutants of residues from the largely movable TM6 located at the interfaces with TM3 (F²⁷²K, F²⁷²Q, and F²⁷²L), TM5 (Y²⁸⁴F), and TM7 (C²⁷⁹A and C²⁷⁹S). Here, we took advantage of mutant rescue by four different chemical classes of pharmacoperones (12) and K¹⁹¹ deletion to reveal previously undetected CA of particular mutants. We used these data to improve the model of the GnRHR in its active state.

Materials and Methods

General

pcDNA3.1 (Invitrogen, San Diego, CA), GnRH analog, D-tert-butyl-Ser⁶-des-Gly¹⁰-Pro⁹-ethylamide-GnRH (Buserelin; Hoechst-Roussel Pharmaceuticals, Somerville, NJ), myo-[2-³H(N)]-inositol (NET-114A; PerkinElmer, Waltham, MA), DMEM, OPTI-MEM, lipofectamine, PBS (GIBCO, Invitrogen), competent cells (Promega, Madison, WI), and Endofree maxi-prep kits (QIAGEN, Valencia, CA), were obtained as indicated.

Mutant receptors

WT and mutant GnRHR cDNA for transfection were prepared as reported (5); the purity and identity of plasmid DNA were verified by dye terminator cycle sequencing (Applied Biosystems, Foster City, CA). GnRHR mutants F²⁷²Q and F²⁷²K were tagged with GFP at the C terminus via a 14-residue spacer (TPSFRADLSRCFCW) derived from the sequence of the catfish GnRHR (24) as previously described (25). Pharmacoperones In3 and Q89 (Merck and Co., Darmstadt, Germany), A177775 (Abbott Laboratories, Abbott Park, IL), and TAK-013 (Takeda Pharmaceuticals, Deerfield, IL) were obtained as indicated (4, 12). The drugs are, respectively, from the following chemical classes: indoles, quinolones (both Q89 and Q103), erythromycin macrolides and (N-{4-[5-{[benzyl(methyl)amino]methyl}-1-(2,6-difluorobenzyl)-2,4-dioxo-3-phenyl-1,2,3,4-tetrahy-drothieno[2,3-d]pyrimidin-6-yl]phenyl}-N′-methoxyurea). Chemical structures (12, 26) and the mechanism of action of these on the hGnRHR (4) have been reported.

Transient transfection

Cos-7 cells were cultured in growth medium [DMEM, 10% fetal calf serum (FCS), and 20 μg/ml gentamicin] at 37 C in a 5% CO₂ humidified atmosphere. For transfection of WT or mutant receptors into cells, 5 × 10⁴ cells were plated in 0.25 ml of growth medium in 48-well Costar cell culture plates. Twenty-four hours after plating, the cells were washed with 0.5 ml of OPTI-MEM and then transfected with WT or mutant receptor DNA with pcDNA3.1 (empty vector) to keep the total DNA constant (100 ng/well). Lipofectamine was used according to the manufacturer's instructions. Five hours after transfection, 0.125 ml of DMEM with 20% FCS and 20 μg/ml gentamicin was added. Twenty-three hours after transfection, the medium was replaced with 0.25 ml of fresh growth medium. Where indicated, pharmacoperones (indicated concentration) in 1% DMSO (vehicle) was added for 4 h in respective media to the cells and then removed 18 h before agonist treatment (8). In the present study, we used trypan blue exclusion to show cell viability after drug exposure.

Inositol phosphate (IP) assays

Twenty-seven hours after transfection, cells were washed twice with 0.50 ml of DMEM, 0.1% BSA, and 20 μg/ml gentamicin and then “preloaded” for 18 h with 0.25 ml of 4 μCi/ml myo-[2-³H(N)]-inositol in inositol-free DMEM, then washed twice with 0.30 ml of DMEM (inositol free) containing 5 mm LiCl and treated for 2 h with 0.25 ml of a saturating concentration of Buserelin (10⁻⁷ m), a metabolically stable GnRHR agonist, in the same medium. When CA was assessed, Buserelin was omitted from the assessment period. Total IP was then determined (27). This assay has been validated as a sensitive measure of plasma membrane expression for functional receptors when expressed at low amounts of DNA (<100 ng/well) and stimulated by excess agonist (1, 6, 8, 11, 28–33).

Binding assays. Saturation and Scatchard analysis

Cells were cultured and plated in growth medium as described above, except 10⁵ cells in 0.5 ml of growth medium were added to 24-well Costar cell culture plates (cell transfection and medium volumes were doubled accordingly). Twenty-three hours after transfection, the medium was replaced with 0.5 ml of fresh growth medium with or without pharmacoperone (1 μg/ml In3). We have reported the details of the Scatchard analysis. Twenty-seven hours after transfection, cells were washed twice with 0.5 ml of DMEM containing 0.1% BSA and 20 μg/ml gentamicin, and then 0.5 ml of DMEM were added. After 18 h, cells were washed twice with 0.5 ml of DMEM, 0.1% BSA, and 10 mm HEPES, then 2 × 10⁶ cpm/ml of [¹²⁵I]-Buserelin, prepared in our laboratory (specific activity, 700–800 μCi/μg; molecular weight, 1363.4), was added to the cells in 0.5 ml of the same medium and allowed to incubate at room temperature for 90 min, consonant with maximum binding (34). New receptor synthesis during this period is negligible at room temperature. After 90 min, the media were removed and radioactivity was measured (35). To determine nonspecific binding, the same concentrations of radioligand were added to similarly transfected cells in the presence of 10 μg/ml unlabeled GnRH.

Confocal microscopy

Cells (10⁵ per well) were plated in 1 ml of DMEM, 10% FCS, and 20 μg/ml gentamicin in Lab-Tek-II Chamber no. 1.5 German Coverglass slides (Nalge Nunc, Naperville, IL) and transfected with 25 ng of GFP-tagged F²⁷²Q or F²⁷²K. Twenty-three hours after transfection, the cells were pretreated with 0.75 μm TAK-013 for 4 h, washed, and incubated for an additional 18 h with DMEM, 0.1% BSA, and gentamicin. ER-Tracker Red (glibenclamide BODIPY TR; Molecular Probes, Eugene, OR) was diluted in DMEM and 0.1% BSA, supplemented with 10 mm HEPES (pH 7.4), to a final concentration of 1 μm and added for 30 min at 37 C. Cells were washed once and then incubated for 10 min with WGA-Alexa Fluor 633 (Molecular Probes), which was diluted to a final concentration of 12.5 μg/ml. After approximately 10 min with the tracking stains, cells were imaged with a Leica SP5 AOBS confocal microscope (Leica Microsystems, Mannheim, GE) using a ×63/1.4 oil objective. ER tracker was excited at 561 nm, and emission was detected in a broad spectral domain from 575- to 630-nm interval. GFP was excited at 488 nm and detected in the 500- to 570-nm interval, and Alexa Fluor 633 was excited at 633 nm and detected at 650–720 nm. GFP and Alexa Fluor 633 were imaged simultaneously; ER tracker was imaged sequentially to eliminate the possibility of bleed through from the GFP channel. Images of single confocal planes were open and contrast enhanced using ImageJ 1.46 (Wayne Rasband; National Institutes of Health, Bethesda, MD).

Data analysis

K_d and B_max (dissociation constant and maximum binding, respectively) values were determined by linear regression analysis using Scatchard plots. Data show means (n ≥ 3); sem are shown. Two-way ANOVA was used as indicated.

Modeling

The homology modeling of hGnRHR was based on crystal structures of the β₂-adrenergic receptor in its inactive state (PDB ID, 2rh1) and in the active conformation bound to Gsα-βγ (PDB ID, 3sn6) as previously described (19). The models can be accessed at http://mosberglab.phar.umich.edu/resources/. The new hGnRHR models were compared with our previous models of hGnRHR (19), built using known crystal structures of the dark-adapted rhodopsin (PDB ID, 1u19) and opsin (PDB ID, 3dqb). The validity of the models was verified by docking of peptide agonists and antagonists into active and inactive conformations, respectively. Ligand docking was performed during distance geometry calculations to satisfy receptor-ligand structural constraints, as described (19).

Results

Radioligand binding of mutants rescued by deletion of residue K¹⁹¹

Specific binding of the radioligand, [¹²⁵I]-Buserelin, was observed in Cos-7 cells expressing WT hGnRHR, and F²⁷²L hGnRHR, but not for mutants F²⁷²K, F²⁷²Q, C²⁷⁹A, C²⁷⁹S, or Y²⁸⁴F. After rescue by pharmacoperone In3, measurable binding of radioligand was detected for F²⁷²K, F²⁷²Q, C²⁷⁹A, C²⁷⁹S, and Y²⁸⁴F. After deletion of residue K¹⁹¹, a modification that increases WT and mutant hGnRHR trafficking to the plasma membrane (10), the maximal amount of radioligand binding is similar to, or higher than, that observed for the WT receptor except for F²⁷²QΔK¹⁹¹. Binding characteristics for WT and mutant receptors was determined after their rescue by In3 (Fig. 1 and Table 1). Consistent with previous observations (36), F²⁷²L showed substantially higher cell-surface expression level, compared with WT receptor. High expression levels were also observed for Y²⁸⁴F, Y²⁸⁴F-ΔK¹⁹¹, C²⁷²A-ΔK¹⁹¹, and C²⁷²S-ΔK¹⁹¹mutants rescued by In3. All mutants, after rescue by In3, demonstrate K_d values that are similar to those of WT receptors with variations that do not exceed 3-fold.

Fig. 1.

Saturation (A) and Scatchard (B) plots of binding of [¹²⁵I]-buserelin to hGnRHR mutants. Cos-7 cells were transfected with 25 ng of DNA for WT receptor or mutant as described in Materials and Methods. After rescue with In3 or by deletion of K¹⁹¹, Scatchard analysis was performed on all mutants as indicated except for C²⁷⁹A, C²⁷⁹S, Y²⁸⁴F, and Y²⁸⁴F-desK¹⁹¹, for which saturation binding was used, in which case the K_d was not determined (nd). The B_max and K_d values are from at least three independent experiments showing the variance between experiments as seen in Table 1. The inset to B shows mutant F²⁷²L that has not been rescued by pharmacoperone In3. The image marked B expanded shows the bottom portion of B, expanded for clarity.

Table 1.

Binding of [¹²⁵I]-Buserelin to the WT and mutant hGnRHR

Mutant	Bmax (fmol/105 cells)	Kd (nm)
WT	7.7 ± 0.9	3.1 ± 1.5
E90K	3.9 ± 0.1	4.6 ± 2.2
E90K-ΔK191	11.0 ± 0.9	2.2 ± 0.4
F272L	32.2 ± 0.5	3.2 ± 0.1
F272K	2.1 ± 0.3	5.6 ± 1.3
F272K-ΔK191	6.8 ± 0.9	2.5 ± 0.4
F272Q	2.3 ± 0.5	4.4 ± 1.0
F272Q-ΔK191	3.3 ± 0.7	1.7 ± 0.6
C279A-ΔK191	14.7 ± 0.6	0.8 ± 0.1
C279S-ΔK191	10.0 ± 1.1	0.9 ± 0.2
C279A	6.7 ± 0.5	nd
C279S	5.0 ± 0.9	nd
Y284F	7.3 ± 0.5	nd
Y284F-ΔK191	15.6 ± 0.5	nd

Cos-7 cells were transfected with 25 ng of receptor or mutant DNA as described in Materials and Methods. After rescue with In3, Scatchard analysis was performed on all mutants as indicated except for C²⁷⁹A, C²⁷⁹S, Y²⁸⁴F, and Y²⁸⁴F-desK¹⁹¹, where saturation binding was utilized and the K_d was not determined (nd). The B_max and K_d values are from at least three experiments showing the mean ± sem.

IC₅₀ of pharmacoperones of the hGnRHR

We next evaluated the ability of four different chemical classes of pharmacoperones (12) to rescue CA. These included an indole (In3), quinolone (Q89), erythromycin macrolide (A177775), and TAK-013. All known pharmacoperones of the GnRHR are also receptor antagonists. The inhibitory potency of these antagonists (IC₅₀) was evaluated by measuring the inhibition of buserelin-stimulated IP production in cells expressing WT and ΔK¹⁹¹-WT hGnRHR (Fig. 2). The IC₅₀ values are, respectively: TAK-013, 5.9 × 10⁻⁹ m; In3, 3 × 10⁻⁸ m; Q89, 2.9 × 10⁻⁸ m; and A177775, 9.0 × 10⁻⁸ m. These values were significantly reduced (by up to 100-fold) in the receptor mutants lacking K¹⁹¹ (Fig. 2B): In3, 9.3 × 10⁻⁸ m; Q89, 1.7 × 10⁻⁷ m; TAK-013, 3.2 × 10⁻⁷ m; and A177775, more than 10⁻⁶ m. The IC₅₀ values of different pharmacoperones to the receptor with K¹⁹¹ deletion correlate with our previous observations for Q89 (19). As we noted before, this effect may be related to the partial occlusion of the ligand binding pocket by the EL2 shifted inside the TM α-bundle after the K¹⁹¹ deletion (19).

Fig. 2.

Antagonist properties of pharmacoperones, In3, Q89, TAK-013, and A17777, assessed by inhibition of buserelin-stimulated IP production. Cos-7 cells were transfected with 10 ng of DNA for (A) hWT or (B) hWT-desK¹⁹¹ GnRHR as described in Materials and Methods. IP production was measured in response to 10⁻⁹ m Buserelin (a metabolically stable GnRH agonist) and in the presence of the indicated level of four different pharmacoperones as indicated. At least three independent experiments were performed in replicates of four to six. The optimal doses of pharmacoperones were selected from earlier studies (4).

CA of mutants rescued by pharmacoperones

We observed that all four pharmacoperones were able to rescue cell-surface expression and stimulated CA in studied mutants but to different extents (Fig. 3A). The effect of pharmacoperones on the rescue of CA mutants generally correlates with their IC₅₀ values (Fig. 2). The highly potent antagonist TAK-013 had the largest rescue effect in most cases, and the least potent A177775 macrolide had the lowest effect on CA. Overall, the highest level of CA was found for F²⁷²K and F²⁷²Q mutants rescued by TAK-013 and In3. These data indicate that the mutants can be functional after reaching the cell surface. WT receptor, which shows no CA, is included as a control, and the horizontal dashed line shows the measured IP background with vector-only transfections; this was not changed with different pharmacoperones. See Supplemental Figs. 1 and 2, published on The Endocrine Society's Journals Online web site at http://mend.endojournals.org, show confocal data that are consistent with the movement of mutants F²⁷²K and F²⁷²Q from the ER (unrescued) to the plasma membrane (rescued by TAK-013).

Fig. 3.

CA of hGnRHR mutants rescued by pharmacoperones and assessed by agonist-independent IP production. IP production was assessed for mutants with K¹⁹¹ (the WT condition) (A) or without K¹⁹¹ (B). Cos-7 cells were transfected with 100 ng of WT (a control that lacks CA) or mutant cDNA as described in Materials and Methods. Mutants were incubated in media alone or rescued with each of four pharmacoperones; these drugs were then washed out, and IP production was measured in response to media alone (no agonist added). In all figures, sem are shown for least three independent experiments performed in replicates of four to six. The horizontal dashed line is IP production after transfection with vector only. Bars marked with an asterisk show values that differ from those obtained after transfection with vector only (two-way ANOVA). DMSO, Dimethylsulfoxide.

CA of mutants rescued by deletion of K¹⁹¹

Figure 3B shows the effect of an alternate method of rescue, the deletion of K¹⁹¹. When K¹⁹¹ was deleted, E⁹⁰K and F²⁷²K mutants demonstrated some CA without pharmacoperone treatment (Fig. 3B). All mutants studied showed higher CA after treatment with at least one pharmacoperone, compared with untreated cells. In the case of mutations at positions 90 and 272, the amount of CA is quite substantial. The observation that K¹⁹¹ deletion alone (i.e. without pharmacoperone rescue) can produce CA suggests that the development of CA is not caused by pharmacoperone drugs and supports the view that each of these approaches enhances trafficking of the mutants to the plasma membrane, and the CA is then measurable.

Stimulated response of mutants rescued by deletion of K¹⁹¹ or by pharmacoperones

As was the case for CA (and expectedly), buserelin-stimulated IP responses were increased by pharmacoperone rescue (Fig. 4A). Deletion of K¹⁹¹ in all studied hGnRHR mutants further enhances the IP production (Fig. 4B). These observations were consonant with increased trafficking to the plasma membrane due to deletion of K¹⁹¹ and/or the use of pharmacoperones. Examination of receptor numbers on the cell surface, assessed with radioactive tracer, [¹²⁵I]-Buserelin, is also consistent with the view that both the pharmacoperone treatment and deletion of K¹⁹¹ were associated with increasing trafficking of the mutants to the plasma membrane (Fig. 1).

Fig. 4.

Agonist-stimulated IP production by mutants of the hGnRHR after pharmacoperone rescue. Functional activity was assessed for mutants with K¹⁹¹ (the WT condition) (A) or without K¹⁹¹ (B). Cos-7 cells were transfected with 10 ng of WT or mutant cDNA, with or without K¹⁹¹ as described in Materials and Methods. After rescue, IP production was measured in response to 10⁻⁷ m buserelin (metabolically stable GnRH agonist). In all figures, sem are shown for least three independent experiments performed in replicates of four to six.

Interestingly, F²⁷²K and F²⁷²Q mutants as well as F²⁷²KΔK¹⁹¹ and F²⁷²QΔK¹⁹¹ mutants demonstrated relatively low levels of buserelin-stimulated IP production that was almost equal to the level of agonist-independent IP produced by these mutants (Figs. 3 and 4). These results suggest that incorporation of polar Q²⁷² or charged K²⁷² likely creates similar distortions at the cytoplasmic end of TM6 as does the agonist binding. An alternative explanation may be related to the inability of these mutants to efficiently bind and respond to Buserelin treatment. However, the improved binding affinity of [¹²⁵I]-Buserelin to these mutants (K_d decreased by 20–50%) (Table 1) made this possibility unlikely.

Modeling of hGnRHR rescued mutants with CA

Our present β₂-adrenoreceptor-based models of the hGnRHR were similar to our previous rhodopsin-based models of hGnRHR within the seven TM α-helices (with ∼2 Å root-mean square deviation) but deviated in the loop regions. In particular, the structures of all intracellular loops and helical ends were corrected in accordance with the β₂-adrenoreceptor template (PDB ID, 3sn6) to provide similar interactions with G protein trimer. In addition, we reconstructed N terminus by building an amphiphilic helix at residues 14–29 and omitting 1–11 region, as functionally unimportant (36). The hydrophobic surface of the amphiphilic helix 14–29 formed by residues C¹⁴, I¹⁷, I²¹, and M²⁴ is facing the ligand-binding pocket. This structure and position of the N terminus may explain the known effect of mutations of C¹⁴ and M²⁴ (10, 11, 13, 36, 37) on ligand binding. The side chain of K¹⁹¹ from the EL2 attached by two disulfides (C¹¹⁴-C¹⁹⁶ and C¹⁴-C²⁰⁰) contacts the N-terminal helix near N¹⁸; glycosylation of this residue is essential for proper receptor trafficking to the plasma membrane (38, 39). The presence of K¹⁹¹ in hGnRHR may preclude N¹⁸ glycosylation in addition to its possible inhibitory effect on the formation of C¹⁴-C²⁰⁰ disulfide (17) or insertion of the EL2 between helices (19). All these factors would impair receptor trafficking to the plasma membrane. The absence of K¹⁹¹ and corresponding steric restrictions in rat and mouse receptors as well as existence of an additional glycosylation site at N⁴ would explain the much more efficient trafficking of rodent receptors to the plasma membrane (10, 11).

Experimental data obtained in the present study also allowed us to validate the improved hGnRHR models. Similarly to the structural templates, our models reproduced all consensus conformational changes associated with transition from the inactive to the active conformation (40–43). The major changes included the movement of TM5 toward TM6, counterclockwise rotation of TM6 (as viewed from the extracellular surface) and the inward shift of TM7 closer to TM2. Such helix rearrangement leads to the contraction of the extracellular ligand binding pocket (Fig. 5) but opened the large intracellular cavity between TM 3–5-6, enabling the insertion of C-terminal helix of Gα in this opening.

Fig. 5.

Location of mutations in the hGnRHR. Helices are shown by cartoons colored blue (inactive conformation) and yellow (active conformation). Substituted residues are shown by sticks, highlighted by dots, and colored purple (inactive conformation) and orange (active conformation). A, Positions of residues mutated in this work (E⁹⁰K, F²⁷²Q, K, C²⁷⁹A, S, Y²⁸⁴F, and ΔK¹⁹¹) in inactive and active conformations of hGnRHR. Side chains of substituted residues (E⁹⁰, F²⁷², C²⁷⁹, and Y²⁸⁴) are shown by balls colored purple (for the inactive state) and orange (for the active state). K¹⁹¹ from the EL2 and proximal N¹⁸ from the N terminus are shown by sticks colored blue. B, Suggested changes in the ligand binding pocket on receptor activation. Positions of mutated residues (C²⁷⁹ and Y²⁸⁴) in the binding pocket of active (yellow) and inactive (blue) receptor conformations. Helix shift and rotamer change of Y²⁸⁴ and C²⁷⁹ during the receptor activation bring Y²⁸⁴ to the nonpolar lipid environment and C²⁷⁹ to the polar environment of the ligand binding pocket. Note the contraction of the ligand binding pocket in the active state caused by the inward movement of TM5, TM3, and TM7. C–J, Interactions between substituted residues in position 272 from the moving TM6 and adjacent residues from TM3, TM5, and TM7 in the model of the inactive and activated hGnRHR. Environment of F²⁷² of hGnRHR (C and G) and its substitutions, F²⁷²L (D and H), F²⁷²Q (E and I), and F²⁷²K (F and J), in inactive (C–F, blue) or active (G–J, yellow) receptor states.

We assumed that structures of G protein-interacting regions should be rather similar in all GPCR. Intracellular ends of seven helices likely undergo similar shifts, which are accompanied by reorientation of residues from conserved GPCR sequence motifs that surround the G protein binding site, such as E/DRY in TM3, PxxxxxxCY in TM5, and D/NPxxY in TM7. On the contrary, rearrangement of residues from the extracellular part of helices, which form dissimilar ligand binding pockets, and from the FxxCWxP motif in the TM6, which acts as a ligand-activated conformational switch, are likely receptor specific and may depend on the ligand type (agonist, partial agonist, antagonist, inverse agonist). Indeed, as it can be noticed from comparison of conformational changes in different GPCR, the movement of the extracellular part of the TM6 was more pronounced during rhodopsin activation caused by cis-trans isomerization of retinal (PDB ID, 1u19 vs. 3pqr) than during agonist-induced activation of β₂-adrenoreceptor (PDB ID, 2rh1 vs. 3sn6). Thus, we expected that identification and modeling of CA mutants would shed light on the conformation of extracellular part of the TM6 in the activated state of hGnRHR.

Indeed, the observed CA of C²⁷⁹S, C²⁷⁹A, and Y²⁸⁴F mutants may indicate that a more polar residue at 279 position and less polar residue at 284 position favor the active state conformation. Therefore, we suggest that during receptor activation, side chains of C²⁷⁹ and Y²⁸⁴ may move together with the whole TM6 or undergo rotamer switch (χ¹ change from −60° to 180°). Either helix or side chain rotation would relocate the C²⁷⁹ side chain inside the water-filled milieu of the ligand binding pocket and Y²⁸⁴ toward the hydrophobic lipid environment (Fig. 5). These conformational changes in the region of the ligand binding pocket are reminiscent to the large TM6 rotation observed in the metarhodopsin II, rather than to the minor TM6 shift seen in the activated β₂-adrenoreceptor.

Data on CA of F²⁷²Q and F²⁷²K mutants (Fig. 3) and on the enhanced folding and cell-surface expression of F²⁷²L mutant (Table 1) confirm the structure of the TM3–TM6 interface in the inactive receptor and large conformational alterations in this region on receptor activation. Comparison of the position of F²⁷² and its substituents in the receptor models showed that this residue is tightly packed in the nonpolar environment of the inactive receptor formed by aliphatic side chains of L⁸⁰, L⁸³, M¹³², I¹³⁵, and I³²² (Fig. 5, A–D, blue). However, F²⁷² becomes more flexible in the more polar environment of the active receptor formed by loosely packed R¹³⁹, N²³¹, Y³²³, M¹³², and M²²⁷ side chains (Fig. 5, E–H, yellow). Aliphatic environment in the inactive receptor is favorable for accommodation of aliphatic L²⁷² and, to a lesser extent, of aromatic F²⁷². Therefore, presence of L²⁷² may promote transition to the inactive receptor conformation and its stabilization. This would explain the higher cell-surface expression of the F²⁷²L mutant and lack of CA (44). On the other hand, presence of polar Q²⁷² and K²⁷² likely destabilizes the compact structure or the inactive conformation and switches it to the more open activated state, where these residues are surrounded by polar groups and are accessible to water molecules. This may explain the observed CA of F²⁷²Q and F²⁷²K mutants.

Discussion

The concept of CA for receptors was introduced in 1989 (45), and it has been theorized that virtually all GPCR have mutants that couple to effectors in the absence of agonist (16). It was curious, in light of the clinical importance of the hGnRHR and the many hundreds of mutants reported for this GPCR, that no mutant with CA was identified among either naturally occurring or designed mutants until recently (17). The present work supports the view that this CA has likely been missed, because the cell recognizes such mutants of the hGnRHR as misfolded and prevents them from being expressed at the plasma membrane.

Mutant E⁹⁰K was the first constitutively active mutant identified for the GnRHR but only after rescue of this protein with pharmacoperones or by deletion of K¹⁹¹ from EL2 (10, 22). In the present study, the approach of revealing undetected CA by rescue was applied to mutants of residues from the movable TM6.

There is a wealth of data, relying on ligand binding and IP production (G protein coupling), that are consistent with the absence of mutants F²⁷²A, E, or K (44), C²⁷⁹A (46), and Y²⁸⁴F (19) at the plasma membrane and a lack of coupling of these mutants to the G protein activation. In contrast, it has been shown that F²⁷²L mutation has increased cell-surface expression (36) without CA (44). We confirmed elevated cell-surface expression of F²⁷²L mutant compared with WT (Fig. 1).

Studying these mutants, we detect CA for F²⁷²Q, F²⁷²K, C²⁷⁹S, C²⁷⁹A, and Y²⁸⁴F, whose trafficking from the ER to the plasma membrane was recovered by pharmacoperone treatment or by deletion of residue K¹⁹¹. The greatest effect was observed for Gln and Lys replacement of the F²⁷² residue from the TM6, which undergoes the large shift from the TM7 toward the TM5 on receptor activation. This may indicate the high structural flexibility of these mutants, which potentially adopt misfolded or activated conformations. The comparable levels of agonist-dependent and agonist-independent IP production by F²⁷²Q and F²⁷²K mutants may indicate that their CA conformation is rather similar to that induced by agonists. The much smaller CA observed for C²⁷⁹A, S, and Y²⁸⁴F mutants coincides with smaller conformational changes in these regions. CA of these mutants may indicate that side chains of C²⁷⁹ and Y²⁸⁴ switch to polar and nonpolar environment, respectively. These observations allowed us to validate the proposed models of the active conformation of hGnRHR.

Previous studies of GPCR revealed CA of some receptors with substitutions at the orthologous position corresponding to F²⁷² of hGnRHR. These include M²⁵⁷Y and M²⁵⁷N mutants of rhodopsin (47, 48), and the L²⁵⁰N, L²⁵⁰Q, L²⁵⁰E, and L²⁵⁰K mutants of melanocortin-4 receptor (49). Naturally occurring L²⁵⁰Q mutation of melanocortin-4 receptor was associated with morbid obesity. In the present studies, we observed CA of F²⁷²K and F²⁷²Q mutants only after their rescue by pharmacoperones or genetic modification. This indicates that the cell protects itself against CA hGnRHR by retaining them in the ER as misfolded proteins.

Like the hGnRHR, other GPCR receptors showed inefficient plasma membrane expression of some WT receptors and numerous mutants that is likely caused by misfolding and misrouting of these proteins (11). Examples include the δ-opioid receptor, V2 vasopressin receptor, GluR1, α₁D adrenoreceptor, odorant, and LH receptors (50–55). It was shown that cell-permeable antagonists, presumably behaving as pharmacoperones, facilitated posttranslational processing and increased export of the ligand-stabilized receptor from the ER to the cell surface of these mutants (5, 55, 56). This suggested that restricted trafficking and the inability to observe CA mutants in the absent of rescue may be a more commonly occurring means of regulating protein availability at the plasma membrane than is presently appreciated and that CA may be revealed for mutants of other receptors by techniques that rescue ER-retained proteins.